In vertebrates, the central nervous system (CNS) consists of two major structural elements, namely, the brain and the spinal cord. The brain in vertebrate species is normally encased within the skull. The spinal cord is continuous with the brain, and in mammals, lies disposed caudally relative to the brain and is encased within and supported by spinal vertebrae. The peripheral nervous system (PNS) comprises nerve cells that lead to and from (and which communicate with cells in) the CNS, often through junctions known as ganglia.
Within the CNS, the main cellular components of the brain and spinal cord are (1) neurons, (2) macroglia, and (3) microglia.
Neurons are the fundamental building blocks of the CNS. They are specialized cells that are electrically excitable, such that they are able to transmit information (which the CNS uses in order to monitor or control numerous body systems and functions) through electrical and chemical signalling methods communicated to receiving neurons, muscle cells or glands.
Structurally, a neuron typically comprises a cell body (which, amongst other structures, contains the cell nucleus, and thus, which stores the cell's genetic information), a main elongated extension from the cell body known as the axon, as well as one or more (and frequently, more than one) branched fine structures that protrude from the cell body, known as dendrites that are capable of forming junctions (known as synapses) with other neurons.
In many CNS neurons, the axon is covered with deposits of a fatty substance known as myelin. Myelin comprises about 40% water, and the dry mass component of the substance comprises about 70-85% lipid and about 15-30% protein. Myelin is deposited in a number of mounds along the length of the axon. When the axon portion of a myelinated neuron is viewed under a microscope, its visual appearance is not dissimilar to that of a string of sausages. On such neurons, myelin is deposited as a tubular coating along the length of the axon in structures that look like mounds, with relatively small spaces (known as “Nodes of Ranvier”) defining a gap between adjoining mounds.
Electrical signals are propagated along the length of a neuron in the following way. Neurons exist in an aqueous extracellular environment that contains electrolytes (most notably, Sodium and Potassium ions). Depolarization and consequent excitation of a neuron in the vicinity of the region that bridges the cell body and the axon results in an influx of Sodium ions across the neuronal cell membrane and into the cell, which initiates an electrical signalling process (known as the “action potential”) that travels along the length of the axon. In an unmyelinated axon, this electrical signal moves constantly as a wave. The function of the myelin coating (known as the “myelin sheath”) in a myelinated neuron is to generate relative regions of electrical insulation between adjoining Nodes of Ranvier (because myelin, comprising fatty substances, acts as an electrical insulator). This means that in turn, the propagation of the action potential along a myelinated axon takes place by the electrical signal “jumping” from one Node of Ranvier to the next. This process, (known as “saltation”) generally accelerates the velocity of the action potential along the length of the axon. Myelin additionally prevents unwanted electrical activity from interfering with the propagation of the action potential along the length of the axon to its end destination.
Many of the neurons in the CNS are myelinated. In normal subjects, myelin is deposited (via a process known as “myelination” or “myelinogenesis”) in multiple tubular coating layers (known as “myelin sheaths”, where—as explained earlier—adjoining sheaths are separated along the length of the axon by the Nodes of Ranvier) located on the axonal region of the neuron. The process of coating axons with myelin, is carried out by specialized macroglia known as “oligodendrocytes”. Oligodendrocytes therefore play a fundamental role of communication and protection in the CNS.
Myelin is considered essential for normal neuronal function in the CNS. Indeed, myelin defects or deficiencies usually result in major neurological problems. Myelin problems can arise as a result of:                (a) processes which interfere with myelination (such processes are generally referred to as “dysmyelination” processes); or        (b) processes which do not interfere with myelination, but instead, which result in an attack on myelin that was previously deposited on neurons in accordance with normal myelinogenesis (these destructive processes are often referred to as “demyelination”).        
Dysmyelination processes (also known as leukodystrophies) often arise from hereditary mutations that affect the synthesis or the formation of myelin. In dysmyelinating conditions, myelin is either abnormally formed or cannot be maintained in its normal state because of an inherited enzymatic or metabolic disorder. One well-known medical condition arising from a dysmyelination process is Allan-Hernon-Dudley Syndrome (or ‘AHDS’). AHDS is a rare X-chromosome linked recessive brain disorder (exclusively seen in males), is characterized by impaired brain development and intellectual disability. Amongst other symptoms, individuals afflicted with AHDS typically exhibit weak muscle tone, impaired muscular development, poor head control and often, a variety of faulty or involuntary movements of the arms and legs. These and other symptoms in AHDS patients typically begin in early childhood. By early adulthood, humans afflicted with AHDS have difficulty in walking independently, and many end up being wheelchair bound. AHDS is a progressive disorder.
AHDS is caused by mutations in the SLC16A2 (solute carrier family 16A2) gene. The SLC16A2 gene encodes the genetic instructions for synthesizing a protein, which in turn plays a crucial role in normal CNS development. The protein (known as Monocarboxylate Transporter 8, or MCT8) transports an endogenous hormone (triiodothyronine [also known as T3]) into neurons and glial cells in the developing brain. T3 is produced and secreted by the thyroid gland. T3 is known to be critical for the normal brain development, including normal myelination and growth of neurons and glial cells, as well as the establishment of neuronal synapses.
Demyelination usually results from certain neurodegenerative disorders. The most widely known condition of this nature is multiple sclerosis (MS). Although the exact cause of MS presently remains unknown, it is currently believed that in most instances, MS is an autoimmune-like disorder, and that in at least some forms of MS, exposure of a subject to an extrinsic pathogen (such as a bacterium or virus) or a chemical agent may be involved in its onset. As part of the autoimmune response, inflammatory cells invade the CNS, causing damage to the brain, spinal cord, and/or the optic nerves. In particular, the inflammatory cells target and damage the protective myelin sheath that coats myelinated neurons. This damage causes the formation of scars (called ‘plaques’ or ‘lesions’) on the affected neuronal tissue, which interfere with the normal processes of neuronal transmission.
The clinical symptoms exhibited by subjects afflicted with MS can be significant, and typically include the following (amongst others):                (a) fatigue, which often manifests as a feeling of debilitation that is disproportionate to an activity in which the subject is engaged;        (b) balance and co-ordination problems;        (c) pain;        (d) speech abnormalities;        (e) psychological or emotional disturbances; and        (f) blindness.        
There is currently no known cure for either dysmyelinating or demyelinating conditions. In particular, there are presently no therapies known which would address the myelin defects or deficiencies that cause, or are involved in the development of these conditions, or of other conditions (eg, spinal cord injury), where remyelination or repair/replacement of damaged myelin would be highly desirable, if that were possible. Specifically, re-myelination (ie, “myelin repair” or “myelin replacement”) is not a possibility on the current state of scientific knowledge. In humans, myelination begins in the third trimester of gestation, and the overwhelming majority of the myelination process is therefore completed by the adolescent years. In subjects who either suffer spinal cord injury (SCI) or who suffer from a dysmyelinating or demyelinating condition, the prevailing wisdom has been that the state of medical knowledge therefore offers little if any realistic hope for cures to these degenerative conditions.
In the last decade or so, stem cell science has offered new hope for the treatment of some conditions that were previously considered untreatable. In the context of dysmyelinating and demyelinating conditions in humans, it has been postulated that if human oligodendrocyte precursor cells (hOPC) could be generated from human embryonic stem cells (hESC), hOPCs generated in this manner could be used as part of a cell replacement therapy (CRT) in humans who suffer from either a dysmyelinating or a demyelinating condition.
hESCs are derived from the inner cell mass of the blastocyst from the pre-implantation stage of the human embryo, and are “pluripotent” (Thompson et al., 1998), meaning that they can differentiate into any cell type derived from the three primary germ layers: ectoderm, mesoderm and endoderm. Each corresponding germ layer has the potential to differentiate into different compartments of the body. Ectoderm derivatives include neural and epithelial lineage cells.
Despite the optimism pertaining to the use of hESCs as a potential source of large numbers of exogenous OPCs for CRT, to date an established protocol for generating acceptably homogenous populations of OPCs that are able to myelinate efficiently does not exist. An established protocol to generate pure populations of hESC-derived oligodendrocytes would advance the quality of any future clinical trials of CRT in conditions involving dysmyelination or demyelination. This would provide a potential therapeutic strategy for individuals (male or female) suffering from delayed myelination conditions, such as AHDS, Pelizaeus-Merzbacher disease (PMD), Canavan disease and Alexander disease (amongst other leukodystrophies). Importantly, the possibility for therapeutic interventions also exists for acute and chronic demyelination, such as occurs in MS and SCI.
hOPCs can be experimentally derived from hESCs in culture in the presence of specific growth factor-defined conditions in a culture medium. The desired outcome of hESC-derived oligodendrocyte differentiation is to obtain a sufficient yield of acceptably homogeneous populations of OPCs to be utilized for potential CRT. However, such an outcome has not yet been achieved, due to current gaps in scientific knowledge and understanding surrounding ex vivo oligodendrogenesis.
Although therapies have in the past been proposed for treating or alleviating some dysmyelinating and demyelinating conditions by using certain thyroid hormones or their analogues, the biological mechanisms responsible for the genesis and onset of such conditions have hitherto not been well understood, and hence, the previously attempted treatments are unlikely to have been as effective as they could be. Accordingly, and while a number of approaches have been used to date to use thyroid hormones or their analogues to treat such conditions, an inadequate understanding of the responsible biological mechanisms at play has hampered the development of suitable or more effective therapies for those conditions.
The present invention therefore aims to alleviate at least one of these problems, and to provide improved methods of generating acceptably homogeneous populations of OPCs for use in research or potentially for use in CRT, as well as aiming to provide methods of treating dysmyelinating and/or demyelinating conditions that are based on an improved understanding of the biological mechanisms responsible for causing them.